Immune responses of hemocytes in the blood clam Tegillarca granosa in response to in vivo Vibrio harveyi infection.

Aquaculture of the blood clam Tegillarca granosa accounts for approximately 50% of Arcidae (ark shell) production in China. Vibrio infection severely threatens the sustainability of the clam aquaculture industry. Exposure to Vibrio induces an immune response in blood clams. However, the underlying mechanism remains poorly understood. In this study, immune responses of hemocytes in blood clams were detected after Vibrio infection; the immersion method was used in vivo to mimic the clam's natural infection process. After 24 h of exposure to Vibrio infection, the Vibrio load in hemolymph fluid in both the treatment Ⅰ (25,033.33 ± 19,563.11 CFU/mL) and treatment Ⅱ (122,163.33 ± 194,409.49 CFU/mL) groups were significantly higher, than that in the control group (13.67 ± 37.73 CFU/mL) (P < 0.05). Correspondingly, the production of intracellular reactive oxygen species was approximately 1.40 (treatment Ⅰ) and 2.12 (treatment Ⅱ) fold higher than that in the control group (P < 0.05), and the induced DNA damage showed a similar trend (P < 0.05). Vibrio infection also significantly increased lysozyme content, adenosine triphosphate content, and peroxidase isozyme activity, in both the serum and hemocyte lysates (P < 0.05). The expression of immune-associated genes (ABCA3, c-Myc, Caspase 3, and HSP70) was upregulated under infection conditions. The phagocytic activity was approximately 1.99 (treatment Ⅰ) and 2.57 (treatment Ⅱ) fold that in control clams (P < 0.05). In addition, the total hemocyte count and red granulocyte percentage both significantly decreased by approximately 75-90% after Vibrio infection. These results provided novel insights into the mechanism of hemocyte immunity in T. granosa against Vibrio infection, which may aid in the future prevention and control of Vibrio infection in vivo.

Gamma-aminobutyric acid (GABA) suppresses hemocyte phagocytosis by binding to GABA receptors and modulating corresponding downstream pathways in blood clam, Tegillarca granosa.

Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABAA and GABAB receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl-, which was shown to be leveled down to normal by blocking the ionotropic GABAA receptor. Treatment with GABAA receptor antagonist also rescued the suppression of GABAA receptor-associated protein (GABARAP), KCC, TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), and nuclear factor kappa B subunit 1 (NFκB) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6, IKKα, and NFκB. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABAB receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABAA and GABAB receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates.

Characterization of a novel activating protein-1 (AP-1) gene and the association of its single nucleotide polymorphisms with vibrio resistance in Tegillarca granosa.

The blood clam Tegillarca granosa is a commercial marine bivalve of economic value, accounting for approximately 50% of clam production in China. In recent years, the yield of blood clams has been threatened by bacterial infections caused by marine Vibrio species that thrive under a rising sea temperature. The transcription factor activating protein-1 (AP-1) is emerging as an important player in the innate immunity of marine bivalves against viral or bacterial infections. In this study, the full-length cDNA of a novel T. granosa AP-1 (TgAP-1) was cloned for the first time. The 1591-bp cDNA encoded a protein of 292 amino acid residues with a calculated molecular weight of 32.8 kDa. The TgAP-1 protein contained an N-terminal Jun domain and a C-terminal basic region leucine zipper domain typically found in Jun proteins (a subfamily of AP-1 proteins). TgAP-1 was ubiquitously expressed in T. granosa, with the highest expression detected in the gill and foot, followed by the mantle, hemolymph, and hepatopancreas. Exposure to Vibrio harveyi induced TgAP-1 expression in gill tissues and the expression levels of TgAP-1 of resistant blood clams were always lower than that of control population whether Vibro infection or not. A total of 18 single nucleotide polymorphisms (SNPs) of TgAP-1 were detected in T. granosa. SNP-typing and haplotyping of resistant and susceptible populations revealed that six SNPs (AG type of TgSNP-1, GA type of TgSNP-2, TG type of TgSNP-4, CT type of TgSNP-7, AG type of TgSNP-11, and GA type of TgSNP-12) and four haplotypes (fHap2, fHap3, fHap6, and fHap7) were significantly associated with V. harveyi resistance. Risk assessment showed that fHap2 (CG) and fHap7 (GA) were associated with an increased resistance, while fHap3 (CT) and fHap6 (AG) were associated with an increased susceptibility. The results from this study supported a potential role of TgAp-1 in the anti-Vibro immunity of T. granosa. The discovery of the genetic molecular markers and haplotypes related to Vibrio resistance can provide guidance for selective breeding of T. granosa in the future.

Circadian Rhythm and Neurotransmitters Are Potential Pathways through Which Ocean Acidification and Warming Affect the Metabolism of Thick-Shell Mussels.

Although the impacts of ocean acidification and warming on marine organisms have been increasingly documented, little is known about the affecting mechanism underpinning their interactive impacts on physiological processes such as metabolism. Therefore, the effects of these two stressors on metabolism were investigated in thick-shell mussel Mytilus coruscus in this study. In addition, because metabolism is primarily regulated by circadian rhythm and neurotransmitters, the impacts of acidification and warming on these two regulatory processes were also analyzed. The data obtained demonstrated that the metabolism of mussels (indicated by the clearance rate, oxygen consumption rate, ammonia excretion rate, O:N ratio, ATP content, activity of pyruvate kinase, and expression of metabolism-related genes) were significantly affected by acidification and warming, resulting in a shortage of energy supply (indicated by the in vivo content of ATP). In addition, exposure to acidification and warming led to evident disruption in circadian rhythm (indicated by the heartrate and the expression rhythm of Per2, Cry, and BMAL1) and neurotransmitters (indicated by the activity of acetyl cholinesterase and in vivo contents of ACh, GABA, and DA). These findings suggest that circadian rhythms and neurotransmitters might be potential routes through which acidification and warming interactively affect the metabolism of mussels.

Expression of ABCA3 transporter gene in Tegillarca granosa and its association with cadmium accumulation.

Exposure to cadmium (Cd), a heavy metal, can cause strong and toxic side effects. Cd can enter the body of organisms in several ways, leading to various pathological reactions in the body. Tegillarca granosa is a kind of bivalve shellfish favored by people in the coastal areas of China. Bivalve shellfish can easily absorb heavy metal pollutants from water bodies while filter feeding. T. granosa is considered a hyper-accumulator of Cd, and the TgABCA3 gene is highly expressed in individuals with a high content of Cd-exposed blood clam. However, it is unclear whether TgABCA3 is involved in Cd ion transport in blood clam and the molecular mechanism for the mechanism of the Cd-induced responses for maintaining cell homeostasis. In this study, the complete cDNA of the TgABCA3 gene was analyzed to provide insights into the roles of TgABCA3 in resistance against Cd in blood clam. The complete sequence of TgABCA3 showed high identity to that of TgABCA3 from other bivalves and contained some classical motifs of ATP-binding cassette transport proteins. TgABCA3 expression in different tissues was measured using real-time quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. The tissue-specific expression showed that TgABCA3 expression was highest in the gill tissue. The TgABCA3 expression in the gill tissue was silenced using the RNA interference technique. After TgABCA3 silencing, the TgABCA3 expression decreased, the Cd content increased, the oxygen consumption and ammonia excretion rates increased, and the ingestion rate decreased. These results showing that the extents of Cd accumulation and resulting toxic effects are related to expression levels and activity of TgABCA3 indicate that TgABCA3 has a protective function against Cd in the clam. This increase in Cd accumulation results in serious damage to the body, leading to the enhancement of its physiological metabolism. Therefore, the findings of the study demonstrated that TgABCA3 can participate in the transport of Cd ions in the blood clam through active transport and play a vital role in Cd detoxification.

Molecular cloning, characterization, and tissue distribution of c-Myc from blood clam Tegillarca granosa and its role in cadmium-induced stress response.

Cadmium (Cd) pollution threatens the cultivation of the blood clam Tegillarca granosa (T. granosa) in coastal regions of the East China Sea. The molecular mechanisms regulating Cd stress response and detoxification in blood clams are largely unclear. In the present study, the full-length T. granosa c-Myc (Tgc-Myc) cDNA was cloned for the first time. The 3063-bp cDNA consisted of a 129-bp 5' untranslated region (UTR), a 1746-bp 3' UTR, and a 1188-bp open reading frame encoding a predicted protein of 395 amino acid residues. The predicted protein had a calculated molecular weight of 44.9 kDa and an estimated isoelectric point of 6.82. The predicted protein contained an N-terminal transactivation domain and a C-terminal basic helix-loop-helix leucine zipper domain, which are conserved functional domains of c-Myc proteins. Tgc-Myc showed broad tissue distribution in blood clams, with the highest expression detected in the gill and hepatopancreas. Exposure to Cd, a major heavy metal pollutant in coastal regions of the East China Sea, induced Tgc-Myc expression in gill tissues. Tgc-Myc knockdown led to reduced expression of a variety of stress response/detoxification genes in blood clams cultivated in Cd-contaminated seawater. Tgc-Myc knockdown also led to decreased expression of IGF1R, a proto-oncogene that promotes cell proliferation. These findings indicated that Tgc-Myc regulates Cd-induced stress response and detoxification in blood clams. The upregulation of Tgc-Myc may serve as an approach to generate strains with an enhanced detoxification response and consequently a low heavy metal buildup.

Microplastics boost the accumulation of tetrabromobisphenol A in a commercial clam and elevate corresponding food safety risks.

Marine bivalve molluscs are one of the primary seafood for consumers. Inhabiting terrigenous pollutant-convergent coastal areas and feeding through seawater filtration, edible bivalves are subjected to waterborne emerging pollutants such as microplastics (MPs) and tetrabromobisphenol A (TBBPA). Nevertheless, the potential risks of consuming MP-TBBPA contaminated seafood are still largely unknown. With that, accumulation of TBBPA with and without the presence of MPs in a commercial bivalve species, blood clam (Tegillarca granosa), was determined in the present study. Meanwhile, corresponding target hazard quotients (THQs) as well as margins of exposure (MoEs) were estimated to evaluate the potential health risks for clam consumers. Furthermore, the impacts of pollutants accumulation on the detoxification process and energy supply were analysed. The data obtained demonstrated that MPs aggravate the accumulation of TBBPA in clams, leading to elevated potential food safety risks (indicated by higher THQ values and lower MoE values) for consumers. In addition, the in vivo contents of CYP1A1 and UDP-glucuronosyltransferase, the enzymatic activity of glutathione-S-transferase, and the expression levels of five detoxification-related genes were all dramatically suppressed by MP-TBBPA. Furthermore, clams exposed to MP-TBBPA had significantly lower adenosine triphosphate contents and lower pyruvate kinase and phosphofructokinase activities. These results indicated that the aggravation of TBBPA accumulation may be due to the hence disruption of detoxification process and limited energy available for detoxification.

Transcriptome analysis of sex-related genes in the blood clam Tegillarca granosa.

BACKGROUND: Blood clams (Tegillarca granosa) are one of the most commercial shellfish in China and South Asia with wide distribution in Indo-Pacific tropical to temperate estuaries. However, recent data indicate a decline in the germplasm of this species. Furthermore, the molecular mechanisms underpinning reproductive regulation remain unclear and information regarding genetic diversity is limited. Understanding the reproductive biology of shellfish is important in interpreting their embryology development, reproduction and population structure. Transcriptome sequencing (RNA-seq) rapidly obtains genetic sequence information from almost all transcripts of a particular tissue and currently represents the most prevalent and effective method for constructing genetic expression profiles. RESULTS: Non-reference RNA-seq, an Illumina HiSeq2500 Solexa system, and de novo assembly were used to construct a gonadal expression profile of the blood clam. A total of 63.75 Gb of clean data, with at least 89.46% of Quality30 (Q30), were generated which was then combined into 214,440 transcripts and 125,673 unigenes with a mean length of 1,122.63 and 781.30 base pairs (bp). In total, 27,325 genes were annotated by comparison with public databases. Of these, 2,140 and 2,070 differentially expressed genes (DEGs) were obtained (T05 T08 vs T01 T02 T04, T06 T07 vs T01 T02 T04; in which T01-T04 and T05-T08 represent biological replicates of individual female and male clams, respectively) and classified into two groups according to the evaluation of biological replicates. Then 35 DEGs and 5 sex-related unigenes, in other similar species, were investigated using qRT-PCR, the results of which were confirmed to data arising from RNA-seq. Among the DEGs, sex-related genes were identified, including forkhead box L2 (Foxl2), sex determining region Y-box (Sox), beta-catenin (ß-catenin), chromobox homolog (CBX) and Sex-lethal (Sxl). In addition, 6,283 simple sequence repeats (SSRs) and 614,710 single nucleotide polymorphisms (SNPs) were identified from the RNA-seq results. CONCLUSIONS: This study provided the first complete gonadal transcriptome data for the blood clam and allowed us to search many aspects of gene sequence information, not limited to gender. This data will improve our understanding of the transcriptomics and reproductive biology of the blood clam. Furthermore, molecular markers such as SSRs and SNPs will be useful in the analysis of genetic evolution, bulked segregant analysis (BSA) and genome-wide association studies (GWAS). Our transcriptome data will therefore provide important genetic information for the breeding and conservation of germplasm.